Biomaterial

ABSTRACT

Biomaterial, for example bioactive silicon, may be fabricated by anodizing a silicon wafer to produce a wafer having a porous silicon region. In vitro experiments have shown that certain types of porous silicon cause the deposition of apatite deposits both on the porous silicon and neighboring areas of bulk silicon when immersed in a simulated body fluid solution. This deposition of apatite provides an indication that porous silicon of appropriate form is bioactive, and therefore also biocompatible. A form of porous silicon is dissolved in the simulated body fluid solution and this is an indication of a resorbable biomaterial characteristic. In addition to porous silicon, certain types of polycrystalline silicon exhibit bioactive characteristics. Bioactive silicon may be used in the fabrication of biosensors for in vitro or in vivo applications. The bioactivity of the bioactive silicon may be controlled by the application of an electrical potential thereto.

[0001] The present invention relates to biomaterials.

[0002] A “biomaterial” is a non-living material used in a medical devicewhich is intended to interact with biological systems. Such materialsmay be relatively “bioinert”, “biocompatible”, “bioactive” or“resorbable”, depending on their biological response in vivo.

[0003] Bioactive materials are a class of materials each of which whenin vivo elicits a specific biological response that results in theformation of a bond between living tissue and that material. Bioactivematerials are also referred to as surface reactive biomaterials.Biomaterials may be defined as materials suitable for implantation intoa living organism. L. L. Hench has reviewed biomaterials in a scientificpaper published in Science, Volume 208, May 1980, pages 826-831.Biomaterials which are relatively inert may cause interfacial problemswhen implanted and so considerable research activity has been directedtowards developing materials which are bioactive in order to improve thebiomaterial-tissue interface.

[0004] Known bioactive materials include hydroxyapatite (HA), someglasses and some glass ceramics. Both bioactive glasses and bioactiveglass ceramics form a biologically active layer ofhydroxycarbonateapatite (HCA) when implanted. This layer is equivalentchemically and structurally to the mineral phase in bone and isresponsible for the interfacial bonding between bone and the bioactivematerial. The properties of these bioactive materials are described byL. L. Hench in the Journal of the American Ceramic Society, Volume 74Number 7, 1991, pages 1487-1510. The scientific literature on bioactivematerials often uses the terms HA and HCA on an interchangeable basis.In this patent specification, the materials HA and HCA are collectivelyreferred to as apatite.

[0005] Li et al. have reported the deposition of apatite on silica gelin the Journal of Biomedical Materials Research, Volume 28, 1994, pages7-15. They suggest that a certain density of silanol (SIOH) groups isnecessary to trigger the heterogeneous nucleation of hydroxyapatite. Anapatite layer did not develop on the surface of a silica glass sampleand this is attributed to the lower density of surface silanol groupscompared with silica gel.

[0006] Thick films of apatite have previously been deposited on siliconsingle crystal wafers by placing the wafers in close proximity to aplate of apatite and wollastonite-containing glass dipped into aphysiological solution at 36° C., as described by Wang et al. in theJournal of Materials Science: Materials In Medicine, Volume 6, 1995,pages 94-104. A physiological solution, also known as a simulated bodyfluid (SBF), is a solution containing ion concentrations similar tothose found in the human body and is widely used to mimic the behaviourof the body in in vitro tests of bioactivity. Wang et al. reported thegrowth of apatite on (111) Si wafers but reported that “hardly any”apatite could be grown on (100) Si wafers. The silicon wafer itself isnot bioactive. Wang et al. state that “Si does not play any special rolein the growth of (the) apatite film except that Si atoms on thesubstrate can bond strongly with oxygen atoms in apatite nuclei to forminterfaces with low energy”. The presence of the apatite andwollastonite containing glass is required to induce the deposition ofthe apatite. Indeed, this so-called “biomimetic process” whereby abioactive material is used to treat another material has been shown toinduce apatite growth on a wide variety of bioinert materials, asreported by Y. Abe et al. in the Journal of Materials Science: MaterialsIn Medicine, Volume 1, 1990, pages 233 to 238.

[0007] There is a long felt want for the ability to use silicon basedintegrated circuits within the human body both for diagnostic andtherapeutic purposes. Silicon has been reported to exhibit a poorbiocompatibility in blood, Kanda et al. in Electronics Letters, Volume17, Number 16, 1981, pages 558 and 559, and in order to protectintegrated circuits from damage in biological environments encapsulationby a suitable material is currently required. Medical applications forsilicon based sensors are described in a paper by Engels et al. in theJournal of Physics E: Sci. Instrum., Volume 16, 1983, pages 987 to 994.

[0008] The present invention provides bioactive silicon characterized inthat the silicon is at least partly crystalline.

[0009] Bioactive silicon provides the advantage over other bioactivematerials that it is compatible with silicon based integrated circuittechnol gy. It has the advantag over non-bioactive silicon that itexhibits a greater degree of biocompatibility. In addition, bioactivesilicon may be used for forming a bond to bone or vascular tissue of aliving animal. Bioactive silicon may provide a material suitable for useas a packaging material in miniaturised packaging applications.

[0010] The bioactive nature of the silicon may be demonstrated by theimmersion of the material in a simulated body fluid held at aphysiological temperature, such immersion producing a mineral deposit onthe bioactive silicon. The mineral deposit may be apatite. The apatitedeposit may be continuous over an area greater than 100 μm². Thebioactive silicon may be at least partially porous silicon. The poroussilicon may have a porosity greater than 4% and less than 70%.

[0011] Bulk crystalline silicon can be rendered porous by partialelectrochemical dissolution in hydrofluoric acid based solutions, asdescribed in U.S. Pat. No. 5,348,618. This etching process generates asilicon structure that retains the crystallinity and thecrystallographic orientation of the original bulk material. The poroussilicon thus formed is a form of crystalline silicon. At low levels ofporosity, for example less than 20%, the electronic properties of theporous silicon resemble those of bulk crystalline silicon.

[0012] Porous silicon may be subdivided according to the nature of theporosity. Microporous silicon contains pores having a diameter less than20 Å; mesoporous silicon contains pores having a diameter in the range20 Å to 500 Å; and macroporous silicon contains pores having a diametergreater than 500 Å. The bioactive silicon may comprise porous siliconwhich is either microporous or mesoporous.

[0013] Silicon has never been judged a promising biomaterial, incontrast with numerous metals, ceramics and polymers, and has never beenjudged capable of exhibiting bioactive behaviour. Indeed, nosemiconductors have been reported to be bioactive. Silicon is at bestreported to be relatively bioinert but gen rally exhibits poorbiocompatibility. Despite the advances made in miniaturisation ofintegrated circuitry, silicon VLSI technology is still under developmentfor invasive medical and biosensing applications, as described by K. D.Wise et al. in “VLSI in Medicine” edited by N. G. Einspruch et al.,Academic Press, New York, 1989, Chapter 10 and M. Madou et al. in Appl.Biochem. Biotechn., Volume 41, 1993, pages 109-128.

[0014] The use of silicon structures for biological applications isknown. International patent application PCT/US95/02752 having anInternational Publication Number WO 95/24472 describes a capsule havingend faces formed from a perforated amorphous silicon structure, whosepores are large enough to allow desired molecular products through butwhich block the passage of larger immunological molecules, to provideimmunological isolation of cells contained therein. No evidence as tothe biocompatibility of the silicon structure is provided, and workersskilled in the field of biocompatible materials would expect that such adevice would in vivo stimulate the production of fibrous tissue whichwould block the pores. It is known that when micromachined siliconstructures are used as sensors for neural elements a layer of fibroustissue forms between the silicon surfaces and the neural elements ofinterest, as reported by D. J. Edell et al. in IEEE Transactions onBiomedical Engineering, Volume 39, Number 6, 1992 page 635. Indeed thethickness and nature of any fibrous issue layer formed is often used asone measure of biocompatibility, with a thinner layer containing littlecell necrosis reflecting a higher degree of biocompatibility.

[0015] U.S. Pat. No. 5,225,374 describes the use of porous silicon as asubstrate for a protein-lipid film which interacts with target speciesto produce an electrical current when exposed to target species in an invitro solution. The porous silicon is oxidised to produce a hydrophilicsurface and is chosen since the pores act as a conduit for anion-current flow and the structure provides structural support for thelipid layer. The porous silicon is separated from the in vitro solutionby the protein-lipid film and so the question of the bioactivity orbiocompatibility of the porous silicon does not arise.

[0016] Porous silicon has been suggested as a substrate material for invitro biosensors by M. Thust et al. in Meas. Sci. Technol, Volume 7 1996pages 26-29. In the device structure described therein, the poroussilicon is subjected to a thermal oxidation process to form a silicondioxid lay r on the exposed silicon surfaces of the pores.

[0017] Since the porous silicon is partially thermally oxidised, thebioactivity or biocompatibility of the silicon is not of relevance sinceit is only the silicon dioxide which is exposed to test solutions. Theporous silicon is effectively an inert host for enzyme solutions.

[0018] Microperforated silicon membranes have been described as beingcapable of supporting cell structures by E. Richter et al. in Journal ofMaterials Scienc : Materials in Medicine, Volume 7, 1996, pages 85-97,and by G. Fuhr et al. in Journal of Micromechanics and Microengineering,Volume 5, Number 2, 1995, pages 77-85. The silicon membranes describedtherein comprises silicon membranes of thickness 3 μm perforated bysquare pores of width 5 μm to 20 μm using a lithography process. Mouseembryo fibroblasts were able to grow on cleaned membranes but adherenceof the cells was improved if the membranes were coated with polylysine.This paper is silent as to the bioactivity of the silicon membrane, andthere is no mention of an apatite layer having been formed when exposedto the cell culture medium. Indeed, given the dimensions of the poresused, the structure is not likely to exhibit a significant degree ofbioactivity. Furthermore, it is accepted by Fuhr et al. that there isstill a need to find and develop cell-compatible materials with longterm stability.

[0019] A. Offenhäusser et al. in Journal of Vacuum Science Technology A,Volume 13, Number 5, 1995, pages 2606-2612 describe techniques forachieving biocompatibility with silicon substrates by coating thesubstrate with an ultrathin polymer film. Similarly, R. S. Potember etal. in Proc. 16th Int. Conf. IEEE Engineering in Medicine and BiologySociety, Volume 2, 1994, pages 842-843 describe the use of a syntheticpeptide attached to a silicon surface to promote the development of ratneurons.

[0020] In a further aspect, the invention provides a bioactive siliconstructure characterized in that the silicon is at least partlycrystalline.

[0021] In a still further aspect, the invention provides an electronicdevice for operation within a living human or animal body, characterizedin that the device includes bioactive silicon.

[0022] Bioactive silicon of the invention may be arranged as aprotective covering for an electronic circuit as well as a means forattaching a device to bone or other tissue.

[0023] The electronic device may be a sensor device or a device forintelligent drug delivery or a prosthetic device.

[0024] In a still further aspect, the invention provides a method ofmaking silicon bioactive wherein the method comprises making at leastpart of the silicon porous.

[0025] In another aspect, the invention provides a method of fabricatingbioactive silicon, characterized in that the method comprises the stepof depositing a layer of polycrystalline silicon.

[0026] In a yet further aspect, the invention provides biocompatiblesilicon characterized in that the silicon is at least partlycrystalline.

[0027] In a still further aspect, the invention provides resorbablesilicon.

[0028] In another aspect, the invention provides a method ofaccelerating or retarding the rate of deposition of a mineral deposit onsilicon in a physiological electrolyte wherein the method comprises theapplication of an electrical bias to the silicon.

[0029] The silicon may be porous silicon.

[0030] In a further aspect, the invention provides bioactive materialcharacterised in that the bioactivity of the material is controllable bythe application of an electrical bias to the material.

[0031] Conventional bioactive ceramics are electrically insulating andtherefore preclude their use in electrochemical applications. Where theelectrical simulation of tissue growth has been studied previously, ithas often been difficult to distinguish the direct effects of electricfields from those associated with an altered body chemistry nearimplanted “bioinert” electrodes.

[0032] In a still further aspect, the invention provides a compositestructure comprising bioactive silicon region and a mineral depositthereon characterized in that the silicon region comprises silicon whichis at least partly crystalline.

[0033] A possible application of the invention is as a substrate forperforming bioassays. It is desirable to be able to perform certaintests on pharmaceutical compounds without resorting to performing testson living animals. There has therefore been a considerable amount ofresearch activity devoted to developing in vitro tests in which celllines are supported on a substrate and the effects of pharmaceuticalcompounds on the cell lines monitored. A composite structure of siliconand apatite might provide a suitable substrate for such tests.

[0034] In a further aspect, the invention provides a method offabricating a biosensor, characterized in that the method includes thestep of forming a composite structure of bioactive silicon and a mineraldeposit thereon.

[0035] The invention further provides a biosensor for testing thepharmacological activity of compounds including a silicon substrate,characterized in that at least part of the silicon substrate iscomprised of bioactive silicon.

[0036] In order that the invention may be more fully understood,embodiments thereof will now be described, by way of example only, withreference to the accompanying drawings, in which:—

[0037]FIG. 1 is a schematic sectional diagram of a bioactive siliconwafer;

[0038]FIG. 2 is a representation of a scanning electron microscope (SEM)micrograph of an apatite deposit on a bulk silicon region adjacent aporous region of the FIG. 1 wafer;

[0039]FIG. 3 is a representation of an SEM micrograph of a cross-sectionof the FIG. 2 silicon region;

[0040]FIG. 4 is a representation of an SEM micrograph showing an apatitespherulite deposited on a porous silicon region of porosity 31%

[0041]FIG. 5a is a representation of an SEM micrograph of an unanodisedregion of a silicon wafer anodised to produce a porosity of 48% afterimmersion in a simulated body fluid solution;

[0042]FIG. 5b is a representation of an SEM micrograph of an anodisedregion of the FIG. 5a wafer;

[0043]FIG. 6 is a schematic diagram of a biosensor incorporatingbioactive silicon;

[0044]FIG. 7 is a schematic diagram of an electrochemical cell for theelectrical control of bioactivity;

[0045]FIG. 8 is a plot of a calcium concentration profile in poroussilicon wafers after treatment in the FIG. 7 cell; and

[0046]FIG. 9 is a schematic diagram of a biosensor device incorporatingbioactive polycrystalline silicon of the invention.

[0047] Referring to FIG. 1 there is shown a section of a bioactivesilicon wafer, indicated generally by 10. The silicon wafer 10 comprisesa porous silicon region 20 and a non-porous bulk silicon region 22. Theporous region 20 has a thickness d of 13.7 μm and an average porosity of18%. The silicon wafer 10 has a diameter l of three inches or 75 mm. Theporous region 20 has a surface area per unit mass of material of 67 m²g⁻¹. This was measured using a BET gas analysis technique, as describedin “Adsorption, Surface Area and Porosity” by S. J. Gregg and K. S. W.Sing, 2nd edition, Academic Press, 1982.

[0048] The wafer 10 was fabricated by the anodisation of a heavilyarsenic doped Czochralski-grown (CZ) n-type (100) silicon wafer havingan initial resistivity of 0.012 Ωcm. The anodisation was carried out inan electrochemical cell, as described in U.S. Pat. No. 5,348,618,containing an electrolyte of 50 wt % aqueous HF. The waf r was anodisedusing an anodisation current density of 100 mAcm⁻² for one minute. Thewafer was held in place in the electrochemical cell by a syntheticrubber washer around the outside of the wafer. Consequently, an outerring of the wafer remained unanodised after the anodisation process.This outer unanodised ring is shown in FIG. 1 as a non-porous bulksilicon region 22. The unanodised ring has a width s of 4 mm.

[0049] In order to determine the bioactivity of anodised wafers, cleavedwafer segments were placed in a simulated body fluid (SBF) solution fora period of time ranging from 2 hours to 6 weeks. The SBF solution wasprepared by dissolving reagent grade salts in deionised water. Thesolution contained ion concentrations similar to those found in humanblood plasma. The SBF solution ion concentrations and those of humanblood plasma are shown at Table 1. The SBF solution was organicallybuffered at a pH of 7.30±0.05, equivalent to the physiological pH, withtrihydroxymethylaminomethane and hydrochloric acid. The porous waferswere stored in ambient air for at least several months prior toimmersion in the SBF solution and were therefore hydrated porous siliconwafers. The porous silicon thus comprised a silicon skeleton coated in athin native oxide, similar to that formed on bulk silicon as a result ofstorage in air. TABLE 1 Concentration (mM) Ion Simulated Body FluidHuman Plasma Na⁺ 142.0 142.0 K⁺ 5.0 5.0 Mg²⁺ 1.5 1.5 Ca²⁺ 2.5 2.5 HCO₃ ⁻4.2 27.0 HPO₄ ²⁻ 1.0 1.0 Cl⁻ 147.8 103.0 SO₄ ²⁻ 0.5 0.5

[0050] Cleaved wafer segments having typical dimensions of 0.4×50×20 mm³were placed in 30 cm³ capacity polyethylene bottles filled with the SBFsolution and held at 37°±1° C. by a calibrated water bath.

[0051] After a known period of tim , the segments were removed from theSBF solution, rinsed in deionised water and allowed to dry in ambientair prior to characterisation. The SBF treated segments were examinedusing scanning electron microscopy (SEM) and x-ray microanalysis (EDX)on a JEOL 6400F microscope. Secondary ion mass spectrometry was carriedout using a Cameca 4F instrument and infrared spectroscopy was performedusing a Biorad FTS-40 spectrometer.

[0052] After periods of immersion in the SBF solution of 2, 4, and 17hours, there were negligible apatite deposits on both the porous siliconregion 20 and the non-porous bulk silicon region 22.

[0053] Referring to FIG. 2 there is shown a reproduction of an SEMmicrograph indicated generally by 50. The micrograph 50 is an image ofpart of the region 22 after the wafer 10 had been placed in the SBFsolution for a period of 6 days. A scale bar 52 indicates a dimension of2 μm. The micrograph 50 shows a continuous layer of apatite spherulites54 covering the surface of the region 22. The apatite spherulites hadnucleated at a sufficiently high density to create a relatively smoothfilm in which boundaries between spherulites such as boundary 56 areindistinct The film was continuous over an area of at least 100 μm².

[0054] Referring to FIG. 3 there is shown a reproduction of an SEMmicrograph, indicated generally by 100, of a cross-section of the wafer10 in the region 22 after the wafer had been immersed in the SBFsolution for 6 days. A scale bar 102 indicates a dimension of 1.0 μm.The micrograph 100 indicates three distinct regions, indicated by theletters A, B, and C. EDX analysis confirmed that region A is silicon,corresponding to the original material of the non-porous bulk siliconregion 22. Region B exhibited both silicon and oxygen peaks under EDXanalysis, indicating that region B comprises silicon oxide. Region Cexhibited calcium, phosphorus and oxygen peaks under EDX analysis,consistent with this region comprising spherulites of apatite. Thecombined SEM and EDX analysis demonstrates that a porous silicon oxidelayer (region B) has formed on the bulk silicon (region A), therebyenabling nucleation and coverage with apatite (region C).

[0055] SEM analysis of the wafer 10 in the area of the porous siliconregion 20 after 6 days immersion in th SEF solution indicated a muchlower l vel of apatite coverage compared with the region 22. The poroussilicon region 20 contains a high level of mesoporosity. After 10 daysimmersion in the SBF solution in which significant layer erosion of theporous silicon had occurred, macropores were visible under SEM analysisin the region 20. The combined SEM and EDX analysis demonstrates that,in contrast to the bulk silicon region 22, apatite nucleation can occurdirectly on the porous silicon region 20 and does not require theformation of an intermediate porous silicon oxide layer. The intentionalintroduction of very large (greater than 100 μm diameter) macropores maybe advantageous in that it may enable vascular tissue to grow within thestructure of the porous silicon.

[0056] The formation of apatite deposits has also been observed onwafers having porous silicon porosities other than 18%. A microporouswafer having a porous silicon region with a porosity of 31% wasfabricated from a 0.03 Ωcm heavily boron doped p-type CZ silicon waferby anodisation at an anodisation current density of 100 mAcm⁻² for oneminute in 50 wt % HF. The resulting porous silicon region had athickness of 9.4 μm and a surface area per unit mass of 250 m² g⁻¹. Theporous silicon wafer was heavily aged prior to immersion in the SBFsolution.

[0057]FIG. 4 shows a representation of an SEM micrograph, indicatedgenerally by 150, of the surface of the 31% porosity porous siliconlayer after a segment of the wafer had been immersed in 30 cm³ of theSBF solution for 7 days. The micrograph 150 shows spherulites such as aspherulite 152 of apatite on the surface 154 of the porous silicon.

[0058] Microporous wafers having a porous silicon region of a porosityof 48% were fabricated by anodising a lightly boron doped p-type siliconwafer having a resistivity of 30 Ωcm in 50 wt % HF at an anodisationcurrent density of 20 mAcm⁻² for five minutes. The resulting poroussilicon region had a thickness of 6.65 μm and a surface area per unitmass of approximately 800 m² g⁻¹. The porous silicon wafer segment washeavily aged prior to immersion in a 150 cm³ polyethylene bottle filledwith the SBF solution.

[0059]FIG. 5a shows a representation of a SEM micrograph, indicated gnerally by 200, of an apatit deposit 202 on an unanodised region of the48% porosity wafer after a four week immersion period. FIG. 5b shows arepresentation of a SEM micrograph, indicated generally by 250 of anapatite spherulite 252 deposited on the 48% porosity porous region. Thespherulite 252 exhibits a morphology having a columnar structurecharacteristic of apatite growth on bioactive ceramics as described byP. Li et al. in Journal of Biomedical Materials Research, Volume 28,pages 7-15, 1994. Apatite spherulites having a similar morphology wereobserved on the unanodised region of the wafer. Cross-sectional EDXspectra of the 48% porosity wafer after immersion in the SBF solutiontaken across the unanodised region indicated that spherulites containedcalcium, phosphorus and oxygen, consistent with apatite. Away from thespherulites, an interfacial layer having a thickness of only 150 nmcomprising predominantly silicon and oxygen was observed. Fouriertransform infrared spectroscopy confirms the presence of apatite in boththe porous and non-porous regions. Both the P—O bending vibrationalmodes of P0 ₄ tetrahedra at wavenumbers of around 600 cm⁻¹ and a broadband around 1400 cm⁻¹, attributed to vibrational modes of carbonategroups, were observed.

[0060] Some forms of porous silicon are known to be photoluminescent.The observation of red or orange photoluminescence from porous silicongenerally indicates the presence of quantum wires or quantum dots ofsilicon material. Prior to immersion in the SBF solution, the heavilyaged 48% porosity wafer exhibited photoluminescence, indicating thatdespite being hydrated by exposure to ambient air, the porous siliconregion maintains a high concentration of quantum wires or dots. Theluminescent property was preserved both during and after immersion inthe SBF solution. This shows that apatite may be deposited on poroussilicon such that the luminescent properties are preserved. Preservationof the luminescent properties after growth of an apatite layer may be auseful property for the development of an electro-optical biosensor.

[0061] A wholly mesoporous luminescent porous silicon wafer having a 1μm thick porous region with a porosity of 70% and a surface area perunit mass of 640 m² g⁻¹ was placed in the SBF solution. Afterapproximately one day the porous region had been completely removed bydissolution in the SBF solution and the wafer was no longer lumin sc nt.No apatite deposits were observed on either the porous silicon region orthe non-porous region. It is thought that the mesoporous silicon iswetted more efficiently by the SBF solution and hence the rate ofdissolution is higher for mesoporous silicon than microporous silicon.The mesoporous silicon thus shows resorbable biomaterialcharacteristics. It might be possible to construct a bioactive siliconstructure having a limited area of mesoporous silicon to act as a sourceof soluble silicon. This could produce a locally saturated siliconsolution and hence the promotion of apatite deposition.

[0062] A macroporous silicon wafer having a porous region of 4% porosityand a thickness of 38 μm behaved like a bulk, unanodised silicon waferin as much as it did not exhibit growth of an apatite deposit whenimmersed in the SBF solution for four weeks. In addition, no apatitegrowth has been observed on a porous silicon region having a porosity of80% and a thickness of 50 μm which retains its luminescent propertiesafter two weeks immersion in the SBF solution.

[0063] As a further control, a cleaved non-porous silicon wafer segmentof similar dimensions to the porous silicon wafer segments was placed in30 cm³ of the SBF solution. An extremely low density of micron sizedeposits, less than 5000/cm² was observed after immersion in the SBFsolution for five weeks. These deposits were possibly located at surfacedefects of the silicon wafer. Bulk, non-porous silicon is therefore notbioactive since the rate of growth of apatite deposits is too low for abond to be formed with living tissue.

[0064] These experiments thus indicate that by appropriate control ofpore size and porosity, silicon structures can cover virtually theentire bioactivity spectrum. Bulk and purely macroporous silicon arerelatively bioinert; high porosity mesoporous silicon is resorbable andmicroporous silicon of moderate porosity is bioactive.

[0065] It is known that changes in chemical composition of biomaterialscan also affect whether they are bioinert, resorbable or bioactive. Theabove experiments were carried out on porous silicon wafers which hadnot been int ntionally doped with any specific elements other than theimpurity doping for controlling the semiconductor properties of thesilicon.

[0066] Th elution of calcium from bioactive glass containing SiO₂, Na₂O,CaO and P₂O₅ is believed to significantly assist apatite growth bypromoting local supersaturation. Calcium has been impregnated into afreshly etched layer of microporous silicon of 55% porosity and having athickness of 1.2 μm formed in a lightly doped p-type (30 Ωm) CZ siliconwafer by anodisation at 20 mAcm⁻² for one minute in 40% aqueous HF. Thecalcium impregnation was achieved through mild oxidation by storage in asolution containing 5 g of CaCl.2H₂O in 125 cm³ pure ethanol for 16hours. The impregnation of the porous silicon with calcium, sodium orphosphorus or a combination of these species may promote apatiteformation on silicon.

[0067] The presence of the silicon oxide layer underneath the apatitedeposit at the non-porous region adjacent the porous silicon region ofthe anodised wafers after immersion in the SBF solution indicates thatthe dissolution of silicon from the porous silicon region may be animportant factor for the bioactivity of the porous silicon. Thedissolution of the silicon may form a local supersaturated solutionwhich results in the deposition of a porous silicon oxide layer. Apatiteis then deposited on the porous silicon oxide. This suggests that avariety of non-porous crystalline, polycrystalline or amorphous siliconbased structures containing impregnated calcium and having a highersolubility than normal bulk crystalline silicon in the SBF solution maybe bioactive. To significantly assist apatite growth, the level ofcalcium impregnation needs to be much higher than previously reportedcalcium doped silicon, though the crystallinity of the silicon need notnecessarily be preserved.

[0068] Calcium is generally regarded as an unattractive dopant forsilicon and consequently there have been few studies of calcium dopedsilicon. Sigmund in the Journal of the Electrochemical Society, Volume129, 1982, pages 2809 to 2812, reports that the maximum equilibriumsolubility of calcium in monocrystalline silicon is 6.0×10¹⁸ cm⁻³. Atthis concentration, calcium is unlikely to have any significant effectupon apatite growth. Supersaturated levels of calcium are needed withconcentrations in excess of 10²¹ cm⁻³ (2 at %). Such very highconcentrations may be achieved by:

[0069] (a) solution doping of porous silicon as previously described;

[0070] (b) ion implantation of porous silicon or bulk silicon withcalcium ions; or

[0071] (c) epitaxial deposition of calcium or calcium compounds followedby thermal treatments.

[0072] Referring to FIG. 6 there is shown a schematic diagram of ageneralised sensor, indicated generally by 300, for medical applicationsincorporating bioactive silicon. The sensor 300 comprises two siliconwafer segments 302 and 304. The segment 302 incorporates CMOS circuitry306 and a sensing element 308 linked to the circuitry 306. The sensingelement 308 may be an oxygen sensor, for instance a Clark cell. The CMOScircuitry is powered by a miniaturised battery (not shown) and signalsare produced for external monitoring using standard telemetrytechniques.

[0073] The wafer segment 304 is a micromachined top cover for thesegment 302. The segment 304 has two major cavities 310 and 312 machinedinto it The cavity 310 has a dome shape. When the segments 302 and 304are joined together, the cavity 310 is above the CMOS circuitry 306. Thecavity 312 is circular in cross-section and extends through the segment304 to allow the sensing element 308 to monitor the environmentsurrounding the sensor. The cavity 312 is covered by a permeablemembrane 314. In addition to the major cavities 310 and 312, minorcavities, such as cavities 316, are distributed over a top surface 322of the segment 304. The minor cavities are frusto-conical in shape, withthe diameter of its cross-section increasing into the segment. The minorcavities are present to enable the growth of vascular tissue or bone forbiological fixation. The cavities 310, 312, and 316 are formed bystandard etching techniques, for example ion-beam milling and reactiveion etching through a photoresist mask. At least part of the outersurfaces of the segments 302 and 304 are anodised to form a poroussilicon region in order to promote the deposition of apatite and thebonding of the sensor with the tissue. In FIG. 6, the porous silicon isindicated by rings 330 on the top surface of the segment 304 and grooves332 in the other surfaces. Although FIG. 6 indicates that the outersurfaces of the segments 302 and 304 are covered entirely by poroussilicon, it may be sufficient for only the surface 322 and a bottomsurface 334 of the segment 302 to incorporate porous silicon. Such anarrangement would be simpler to fabricate. The segments 302 and 304 arebonded together using techniqu s d veloped for silicon on insulatortechnologies. Whilst an anodisation technique has been described for theproduction of the porous silicon, stain etching techniques are alsoknown for the production of porous silicon. Such techniques may beadvantageous for producing porous silicon surfaces on complex shapedstructures.

[0074] In addition to sensors, bioactive silicon might find applicationsin electronic prosthetic devices, for example replacement eyes. Otherelectronic devices which may incorporate bioactive silicon might includeintelligent drug delivery systems.

[0075] As well as sensors for incorporation into the bodies of humansand other animals, bioactive porous silicon may be used in thefabrication of biosensors for in vitro applications. A compositestructure of porous silicon with a layer of apatite thereon may haveimproved cell compatibility compared with prior art biosensorarrangements. Biosensors are of potentially great importance in thefield of in vitro pharmaceutical testing. For automated pharmaceuticaltesting, a bioasay device might comprise a silicon wafer having a matrixarray of porous silicon regions. Cells could then be preferentiallylocated at the porous silicon regions and this would facilitateautomated cell analysis after exposure to a pharmaceutical product Theluminescent properties of porous silicon might be utilised to enable anoptical cell analysis technique. Workers skilled in the field ofbiosensors would use their experience to identify which cell cultureswere suitable and how the cells' behaviour could be monitored.

[0076] Whilst the results of in vitro experiments have been described,no in vivo experiments have been described. However, the in vitroexperiments are designed to mimic the environment within a human body.From the results of the in vitro experiments it may be concluded thatthose silicon wafers which produced significant deposits of apatite inthe SBF solution would also exhibit bioactive behaviour in vivo.

[0077] The formation of a film of apatite over a silicon or poroussilicon surface in vitro indicates that the bioactive silicon may be toa certain extent a biocompatible form of silicon. The term“biocompatible” does not necessarily indicate that the material isbiologically acceptable for all applications but that the material isbiologically acceptable for specific applications. Some workers skilledin the field of biocompatibility might regard “tissue compatible” as amore appropriate term to describe this definition of biocompatibility.The lay r of apatite may act as a protective barrier reducing thephysiological effects of the silicon.

[0078] As stated above, mesoporous silicon shows resorbable biomaterialcharacteristics. From the previously referenced paper by Hench in theJournal of the American Ceramic Society, resorbable biomaterials arematerials which are designed to degrade gradually over a period of timeand be replaced by the natural host tissue. The characteristics of themesoporous silicon in the simulated body fluid indicate that mesoporoussilicon of an appropriate porosity may be a resorbable biomaterial. Aspreviously discussed the porous region 20 of the bioactive silicon wafer10 of FIG. 1 contains a high level of mesoporosity. This indicates thatcontrolling the porosity of mesoporous silicon can control whether aporous silicon region is bioactive or resorbable. It may be possible tocontrol the rate at which a porous silicon region is absorbed by tuningthe porosity.

[0079] Although the dissolution of porous silicon in the SBF solutionprovides an indication of resorbable biomaterial characteristics, thebehaviour of a porous silicon region in a living body may be affected byfactors which are not reproducible in the SBF solution. If living cellsgrow on the surface of the porous silicon, these cells may interact withthe porous silicon. Thus experiments carried out in the SBF solution donot provide a clear indication of the suitability of a particular formof porous silicon for resorbable material applications. Experiments mayhave to be carried out in vivo to determine whether a particular desiredphysiological response is achieved.

[0080] Further experiments have been performed which show that it ispossible to either enhance or retard the formation of an apatite layeron the porous silicon by the application of a bias current in the SBFsolution.

[0081] Referring to FIG. 7 there is shown a schematic diagram of anelectrochemical cell 400 for applying a galvanostatic loading to a wholesilicon wafer 402. The wafer 402 is a heavily doped n-type (100)oriented silicon wafer of resistivity 0.012 Ωcm which prior to loadingin the cell 400 was anodized in 40 wt % aqueous HF at 100 mA cm⁻² forone minute to form a bioactive porous silicon layer of approximately 20%porosity having a thickness of 11 μm with a BET measured surface area ofapproximately 70 m² g⁻¹. After anodisation, the wafers are spun dry inair until their weight has stabilised and then immediately loaded intothe cell 400.

[0082] The wafer 402 is inserted into a PTFE cassette 404 and mountedusing a threaded PTFE ring 406 which is screwed into the cassette 404and which compress PTFE coated O-rings 408 and 410. In the cassette 404,the silicon wafer is pushed against a metal back plate 412. The plate412 provides an electrical contact to a rear face of the silicon wafer,and in the cassette an area of 36 cm² of the front porous face of thesilicon wafer is exposed. The cassette 404 is placed in a polycarbonatetank 414, within a waterbath, containing two litres of SBF solutionmaintained at 37±1° C. with organic buffering at pH=7.3±0.05. A spiralplatinum counterelectrode 416 is also inserted into the SBF solution. Ad.c. galvanostatic power supply 418 is used to maintain a constantelectrical current between the wafer 402 and the counterelectrode 416.The wafer 402 may either be under cathodic or anodic bias control. Thepower supply 418 provides a constant current of 36 mA, which correspondsto a current density at the silicon wafer of approximately 1 mA cm² ifcurrent flow is primarily through the silicon skeleton or approximately1 μA cm² if current flow is uniformly distributed across the entiresilicon-SBF interface via the pore network of the porous silicon. Thecurrent flow is maintained for three hours. After removal from the cell400, the wafers 402 are rinsed in deionised water and spun dried.

[0083] After the three hour SBF exposure, the porous silicon wafersurface was examined in a JEOL 6400F scanning electron microscope (SEM)at an accelerating potential of 6 kV. Porosified wafers which wereanodically biased, together with control porosified wafers whichreceived no bias showed no evidence of surface deposits on the poroussilicon. The wafer which was cathodically biased however was completelycovered with spherulites which had merged to form a continuous layer.Plan view EDX analysis showed that this overlayer is a predominantlycalcium and phosphorous containing mineral, with other SBF constituentssuch as carbon, magnesium, sodium and chlorine being close to EDXdetection limits (i.e. <1 atomic %). Plan view EDX analysis of theunbiased and anodically biased wafers showed only the presence ofsilicon and oxygen.

[0084] Cross-sectional SEM and EDX analysis showed that th calcium andphosphorous rich mineral developed under cathodic bias is restricted tothe top of the porous silicon layer and is relatively thin, having athickness of approximately 0.2 μm. Within the porous silicon the calciumand phosphorous levels are below EDX detection limits for all samples.The porous silicon layer given the anodic loading showed a significantbuild up of oxygen within the top 0.5 μm of the layer.

[0085] Secondary ion mass spectrometry (SIMS) was utilised to comparethe extent and depth to which layers were calcified after the threediffering treatments, together with the depth distribution of otherspecific elements. Freshly etched microporous silicon has been shown tocontain very low levels of for example calcium and sodium (present inSBF) but appreciable levels of fluorine (not present in SBF).

[0086]FIG. 8 is a SIMS plot shows the varying levels of calcificationresulting from the electrical biasing treatments. In FIG. 8, the SIMSplot from a cathodically biased wafer is shown by a line 450, the SIMSplot from an unbiased wafer is shown by line 452, and a SIMS plot froman anodically biased wafer is shown by a line 454. Although depositionhas primarily occurred near the surface of the porous silicon, in allcases calcium levels were above the background level throughout the 11μm thick layer. The line 450 shows that cathodic biasing has raised thedegree of calcification and anodic biasing has lowered it compared withthe unbiased wafer. The SIMS measurements also indicated the presence ofthe SBF constituents throughout the porous silicon layer and that therehad been significant movement and loss of fluorine as a result of thecathodic biasing, together with some degree of retention within theoverlayer.

[0087] It is well established that in vitro and in vivo tissues onlyrespond favourably over quite restricted ranges of input power, currentand voltage in electrostimulation experiments. These ranges aresensitive to many factors including the nature of the stimulatingelectrodes. The biasing experiments described above indicate that thekinetics of the calcification process of porous silicon can beaccelerated in vitro and th refore possibly in vivo by the applicationof a cathodic bias. They also suggest that when dissimilar siliconstructures such as porous and bulk silicon are immersed together inphysiological electrolyt s, galvanic corrosion processes may favourcalcification at any cathodic sites that develop.

[0088] The potential applications for the bias control of mineraldeposition are varied. It is known that the insertion of electrodes intoa living organism may result in the formation of a fibrous layer aroundthe electrode, with the thickness of the layer being an indication ofthe biocompatibility of the electrode. The rapid formation of a stablemineral deposit around microelectrodes in vivo offers potential benefitsfor the electrostimulation of tissue growth or the stimulation ofmuscles of paraplegics. The localised control of mineral deposition,where localised regions may be arranged so that a mineral deposit is notformed thereon might have applications in the field of biosensingdevices, both in vivo and in vitro. The process of enhanced mineraldeposition may be beneficial in the coating of silicon based integratedcircuits prior to their implantation in the body.

[0089] Whilst the above description of the electrical control of thedeposition of a mineral is concerned with the deposition on poroussilicon, mineral deposits have also been observed when a cathodic biasis applied to an unanodised wafer in the SBF solution.

[0090] In a further embodiment, it has been found that certain types ofpolycrystalline silicon (polysilicon) are also capable of inducingcalcium phosphate deposition from an SBF solution and are hencebioactive.

[0091] In order to produce bioactive polycrystalline silicon, 100 mmdiameter <100>p-type CZ silicon wafers having a resistivity in the range5 to 10 Ωcm are coated front and back with a 0.5 μm thick wet thermaloxide and subsequently a 1 μm thick polysilicon layer of varyingmicrostructure. The oxide layer is grown in a Thermco TMX9000 diffusionfurnace and the polysilicon layer is grown in a Thermco TMX9000 lowpressure chemical vapour deposition hot walled furnace. For thermaloxide growth, the furnace tube is held at a uniform temperature of 1000°C., and the wet thermal oxid is grown using steam oxidation for 110minutes. Th subsequent deposition of the polysilicon layer involves thepyrolysis of SiH₄ at a pressure in the range 250 to 300 mtorr with thefurnace tube held at a temperature in the range 570 to 620 ° C.

[0092] It is well established that th microstructure of the polysiliconlayer is sensitive to many deposition parameters such as temperature,pressure, gas flow rate, and substrate type, as described in Chapter 2of “Polycrystalline Silicon for Integrated Circuit Applications” by T.Kamins, published by Kluwer Acad. Publ. 1988. Polysilicon layers ofwidely varying microstructure and morphology were obtained by usingdifferent deposition temperatures of 570° C., 580° C., 590° C., 600° C.,610° C., and 620° C. Cross-sectional transmission electron microscopyanalysis revealed that the layer deposited at 570° C. was virtuallyamorphous near its surface whereas the layers deposited at 600° C. and620° C. were polycrystalline throughout their depths. The grain sizevaries appreciably with deposition temperature and significantly withdepth for a given layer.

[0093] Cleaved wafer segments having typical dimensions of 0.5×50×20 mm³were then placed in separate 30 cm³ polyethylene bottles filled with SBFsolution as previously described, with the temperature of the SBFmaintained at 37° C.±1° C. The different polysilicon layers wereobserved to have varying levels of stability in the SBF solution asdetermined by cross-sectional SEM imaging. After 64 hours in the SBFsolution, the polysilicon layer deposited at 620° C. was thinned toapproximately 60% of its original thickness, whereas the thickness ofthe layer deposited at 570° C. was substantially unchanged after 160hours in the SBF solution.

[0094] Mineral deposits were observed to nucleate and proliferate overcertain of the polysilicon layers. These deposits were observed usingplan-view SEM. After two weeks immersion in the SBF solution, mineraldeposits were observed on the polysilicon layers deposited at 600° C.and 620° C. but not on the layer deposited at 570° C. These observationsindicate that as for the porous silicon there is a reactivity window,dependent on the microstructure, for optimum bioactivity. The greatestdensity of mineral deposits were observed with the polysilicon layerdeposited at 600° C. Significant levels of mineral deposits wereobserved on both the front and back of the silicon wafers, consistentwith there having been polysilicon deposition on both sides.

[0095] EDAX analysis of the deposits indicated the presence of calcium,phosphorous and oxygen, consistent with som form of apatite havingnucleated. The morphology of the deposits however differs from that ofthe spherulites previously described in connection with the poroussilicon, with the deposits appearing to be more angular. The reasons forthis are not understood but could reflect a slightly different local pHat the nucleation sites on the polysilicon. P. Li et al. in Journal ofApplied Biomaterials, Volume 4, 1993, page 221, reported that theapatite morphology observed at a pH of 7.3 is significantly differentfrom that observed at a pH of 7.2 for growth on silica gel.

[0096] The potential applications for bioactive polysilicon arepotentially broader than those for bioactive porous silicon. It ispossible to coat a variety of substrates with polysilicon which couldnot be coated with monocrystalline silicon. Surgical implants could becoated with a layer of polysilicon in order to improve adhesion withbone. Polysilicon is also highly compatible with VLSI technologyoffering the prospect of complex electronic circuitry being madebiocompatible. Polysilicon can be surface micromachined in order toproduce a variety of devices and packaging arrangements.

[0097] One possible bioactive silicon packaging concept has already beendescribed with reference to FIG. 6. With bioactive polysilicon, it mightbe possible to construct smaller biochips. Referring to FIG. 9 there isshown a schematic diagram of a biosensor device 500 incorporatingbioactive polysilicon. The device 500 comprises a bulk silicon wafer 510onto which a CMOS circuit 512 and a sensor element 514 are fabricated.The sensor element 514 is electrically connected to the circuit 512. Thecircuit 512 is protected by a barrier layer 516 of for example siliconoxide and silicon nitride. The whole of the device 500 except for awindow 518 to the sensor element 514 is covered with a layer 520 ofbioactive polysilicon. The barrier layer 516 is required becausepolysilicon itself is not a good protective layer for silicon basedcircuitry due to diffusion through grain boundaries. The barrier layer516 is therefore interposed between the circuit 512 and the polysiliconlayer 520.

[0098] By analogy with the results using porous silicon, the bioactivityof polycrystalline silicon might be improved by doping it with calcium,sodium or phosphorus or a combination of these species.

[0099] Bioactive polysilicon might be a suitable substrate for bioassaydevice applications. L. Bousse et al. in IEEE Engineering in Medicineand Biology, 1994 pages 396 to 401 describe a biosensor for performingin vitro measurements in which cells are trapped in micromachinedcavities on a silicon chip. Such an arrangement might beneficiallyincorporate a composite structure of polysilicon with a layer of apatitethereon, the cells locating themselves preferentially on regions ofapatite.

1. Bioactive silicon (20, 520) characterized in that the silicon is atleast partly crystalline.
 2. Bioactive silicon according to claim 1,characterized in that when immersed in a simulated body fluid solutionheld at a physiological temperature the silicon induces the depositionof a mineral deposit (54, C) thereon.
 3. Bioactive silicon according toclaim 2, characterized in that the mineral deposit is apatite. 4.Bioactive silicon according to claim 3, characterized in that theapatite is continuous over at least an area of 100 μm².
 5. Bioactivesilicon according to claim 1, characterized in that the silicon (20) isat least partially porous with a porosity greater than 4% and less than70%.
 6. Bioactive silicon according to claim 5, characterized in thatthe porous silicon is microporous.
 7. Bioactive silicon according toclaim 5, characterized in that the porous silicon is mesoporous. 8.Bioactive silicon according to claim 5, characterized in that the poroussilicon is visibly luminescent.
 9. Bioactive silicon according to claim1 or claim 5, characterized in that the silicon is impregnated with atleast one species taken from a list of calcium, sodium and phosphorus.10. Bioactive silicon according to claim 1, characterized in that thesilicon is polycrystalline silicon (520).
 11. A bioactiv siliconstructure (10, 300, 500) characterized in that th silicon is at leastpartly crystalline.
 12. A bioactive silicon structure according to claim11, characterized in that the structure comprises a porous siliconregion (20) having a porosity greater than 4% and less than 70%.
 13. Abioactive silicon structure according to claim 12, characterized in thatthe porous silicon is microporous.
 14. A bioactive silicon structureaccording to claim 12, characterized in that the porous silicon ismesoporous.
 15. A bioactive silicon structure according to claim 12,characterized in that the structure also includes macropores.
 16. Abioactive silicon structure according to claim 11 or claim 12,characterized in that the silicon is impregnated with at least onespecies taken from a list of calcium, sodium and phosphorus.
 17. Abioactive silicon structure according to claim 16 wherein the poroussilicon is impregnated with calcium at a concentration greater than 10²¹cm⁻³.
 18. A bioactive silicon structure according to claim 11,characterized in that the structure includes resorbable siliconmaterial.
 19. A bioactive silicon structure according to claim 11,characterized in that the structure comprises a region ofpolycrystalline silicon (520).
 20. An electronic device (300, 500) foroperation within a living human or animal body, characterized in thatthe device includes bioactive silicon (20, 520).
 21. An electronicdevice according to claim 20, characterized in that the bioactivesilicon comprises at least partially porous silicon having a porositygreater than 4% and less than 70%.
 22. An electronic device according toclaim 21, characterized in that the porous silicon contains macroporesfor enhancing vascular tissue ingrowth.
 23. An electronic deviceaccording to claim 21, characterized in that the porous silicon extendsat least partially over an outer surface of the device.
 24. Anelectronic device according to any one of claims 20 to 23, characterizedin that the device is a sensor device.
 25. An electronic deviceaccording to claim 20, characterized in that the bioactive silicon ispolycrystalline silicon.
 26. A method of making silicon bioactive, themethod comprising making at least part of the silicon porous.
 27. Amethod according to claim 26, characterized in that the method includesthe impregnation of the porous silicon with calcium.
 28. A method offabricating bioactive silicon, characterized in that the methodcomprises the step of depositing a layer of polycrystalline silicon. 29.The use of bioactive silicon for the construction of a device (300, 500)for use in a living human or animal body characterized in that thesilicon is at least partly crystalline.
 30. Bioactive silicon (20, 520)for use in a method of treatment of the human or animal body. 31.Bioactive silicon (20, 520) incorporated in a device (300, 500) suitablefor use in a living human or animal body characterized in that thesilicon is at least partly crystalline.
 32. Biocompatible silicon (20,520) characterized in that the silicon is at least partly cystalline.33. Biocompatible silicon according to claim 32, characterized in thatwhen immersed in a simulated body fluid solution held at a physiologicaltemperature the silicon induces the deposition of a mineral depositthereon.
 34. Resorbable silicon.
 35. Resorbable silicon according toclaim 34, characterized in that th resorbable silicon comprises a regionof porous silicon such that when immersed in a simulated body fluidsolution the porous silicon dissolves over a period of time.
 36. Amethod of accelerating or retarding the rate of deposition of a mineraldeposit on silicon in a physiological electrolyte wherein the methodcomprises the application of an electrical bias to the silicon.
 37. Amethod according to claim 36, characterized in that the silicon isporous silicon.
 38. Bioactive material (20) characterised in that thebioactivity of the material is controllable by the application of anelectrical bias to the material.
 39. Bioactive electrically conductivematerial (20, 520).
 40. A composite structure (10, 300, 500) comprisingbioactive silicon region (20, 520) and a mineral deposit thereoncharacterized in that the silicon region comprises silicon which is atleast partly crystallin .
 41. A composit structure according to claim40, characterized in that the mineral deposit is apatite.
 42. Acomposite structure according to claim 40 or claim 41, characterized inthat the bioactive silicon region is porous silicon (20).
 43. Acomposite structure according to claim 40 or claim 41, characterized inthat the bioactive silicon is polycrystalline silicon (520).
 44. Amethod of fabricating a biosensor, characterized in that the methodincludes the step of forming a composite structure of bioactive siliconand a mineral deposit thereon.
 45. A biosensor for testing thepharmacological activity of compounds including a silicon substrate,characterized in that at least part of the silicon substrate iscomprised of bioactive silicon.